Immunology 23 November :00

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1 Immunology 23 November :00 Guide for use of these notes First of all thank you for choosing to download these notes to study from I hope you find them useful, please feel free to me if you have any problems with the notes or if you notice any errors. I don't promise to respond to all s but I'll do my best. I organise my notes so that you should read the learning objectives on the left then proceed down the right hand side for a few learning objectives and then cross back over to the left and continue like that. Anything in this highlighted green is a definition or explains basically something's function. Text highlighted in yellow or with a star is what I would deem important and key information. Italics and bold just help to make certain terms stand out. The notes are a bit quirky but I hope you like them and find some of the memory aides strange enough so that they stick in your head. I provide them to you in OneNote format as that is how I created them, they can be saved as PDF but the formatting is not as nice. The one caveat with this is that these notes are freely copy able and editable. I would prefer if you didn't copy and paste my notes into your own but used them as a reference or preferably instead embellished these already existing notes by adding to them. Collection of Immunology Definitions Acute phase proteins- Collection of proteins that initiate the innate immune response. Made up of amyloid phase proteins, mannan binding lectin and c-type reactive protein. Antibodies- Groups of Immunoglobulins present in the blood and body fluids (lymph) that bind to antigens. Antigens- Are molecules that react with antibodies or T cells Cytokines- mall proteins that carry messages between cells. Immunogen- Antigen that engenders an immune response Epitope- ection on an antigen where a receptor binds. Immunopathology- Damage caused to an organism by its own immune system Pathogen associated molecular patterns (PAMPs)- pecific types of molecular structures not normally found in cells Primary immune response: first exposure, antibodies appear after latent period, peak then fall econdary response: greater, quicker, longer response due to memory cells (long lived B-lymphocytes) Good luck with first year tuart Taylor tuart's Immunology Page 1

2 Immunology Overview 27 December :15 Learning Objectives Explain the importance of immunology for human health. Outline the basic principles of immune responses and the timescales in which they occur. Define the terms antigen, antibody, B lymphocyte, T lymphocyte, primary and secondary immune responses, and innate and acquired immunity. Outline the role of clonal selection in immune responses. Understand the role of the physical organization of the immune system in its function. Outline the basic principles of immune responses and the timescales in which they occur. Immune Response Type 1 Interferon α and β Natural Killer Cells Cytotoxic T Lymphocytes Antibody Production Time frame (Days) Peak Type of Immune response Innate Immunity Innate Immunity Acquired Immunity 4-End Increasing Acquired Immunity Explain the importance of immunology for human health. Immunology is the study of the immune system if this goes wrong we can have problems with: 1. Persistent or fatal infection 2. Allergies 3. Transplant rejection 4. Auto-immune disease Role of immune system: To identify harmful pathogens (micro-organisms and toxins) and destroy them, either by distinguishing them from self cells or by recognising danger signals i.e. from inflammation. Immunopathology- Damage caused to an organism by its own immune system Evolutionary arms race between host's immune system and the pathogen. Pathogen has much quicker generation time and thus can evolve faster, therefore we rely on a flexible and adaptive immune response. Define the terms antigen, antibody, B lymphocyte, T lymphocyte, primary and secondary immune responses, and innate and acquired immunity. Primary Immune Response- low response after an antigen is encountered for the first time. econdary Immune Response- Quicker response the second time an antigen is encountered. This is due to the creation of memory cells- lymphocytes already primed to destroy the antigen. B lymphocyte- A type of white blood cell concerned with the production of antibodies via plasma cells, antigen presentation and formation of memory cells. T-lymphocytes- Leukocytes who's function is to be cytotoxic and kill infected cells and to activate and direct other immune cells. Innate Immunity Depends on preformed cells and molecules Fast starts in hours Limited specificity- pattern recognition of danger signals Acquired Immunity Depends on clonal selection. Development of cells that are specific to antigen low starts in days Highly specific Antibodies- Groups of Immunoglobulins present in the blood and body fluids (lymph) that bind to antigens. Innate Immunity Anatomical barriers- Waterproof epidermis restricts entrance to pathogens, mucus and cilia within respiratory system. Physiological barriers- Low ph in the stomach kills bacteria. Complement system, interferons, lysozyme and anti-microbial peptides. Functions of the innate immune system: to buy time whilst the adaptive immune system fashions a specific response stimulates adaptive immune system through complement and cytokines. Detection of viruses Pathogen associated molecular patterns (PAMPs)- pecific types of molecular structures not normally found in cells Examples include dsrna (double stranded RNA) and certain carbohydrates e.g. mannose -> mannose binding lectin complement pathway. PAMPs bind to cell receptors: 1. Toll-like receptors (TLR)- cell surface intracellular 2. RIG-I-like receptors (RLR)- cytoplasmic 3. Nucleotide-binding domain, leu-rich repeat-containing proteins (NLR) 4. C-type lectin receptors (CLR) Adaptive Immunity Function- To destroy invading pathogens in a precise manner by responding to and targeting specific proteins. Cellular Immunity- B and T lymphocytes Humoral Immunity- Antibodies Antibodies- Groups of Immunoglobulins present in the blood and body fluids (lymph) that bind to antigens. Antigens- Are molecules that react with antibodies or T cells Receptors elicits a signalling cascade, resulting in anti-viral responses Interferon types 1 and 3 ---> enzymes that degrade viral nucleic acid & retroviral restriction factors Acute phase inflammatory response Innate Immune Response Occurs as a result of tissue damage. Rise in body temperature coupled with increased production of a number of proteins by the liver: C-reactive protein tuart's Immunology Page 2

3 Antibodies- Groups of Immunoglobulins present in the blood and body fluids (lymph) that bind to antigens. Antigens- Are molecules that react with antibodies or T cells Immunogen- Antigen that engenders an immune response Five types of Immunoglobulins- covered in antibodies lecture. How antibodies kill viruses: Four important mechanisms: 1) binds to virus and prevents attachment to cell. 2) opsonisation: virus-ab complex is phagocytosed by MΦ. 3) complement-mediated lysis of enveloped viruses. 4) antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by natural killer (NK)-like cells. Agranular leukocytes % of WBC circulating. 99% of all cells in lymph. Lymphocytes B lymphocytes- Antibody producing cells Natural Killer cells T- lymphocytes- 1) Cytotoxic Lymphocytes (CTL) CD8+ 2) Helper T Cells CD4+ Lymphocyte Antigen Receptors B lymphocytes have a membrane bound antibody receptor that binds intact antigens whereas T lymphocytes receptor has a α and β chain that creates the T Cell antigen Receptor (TCR) which binds digested antigens. Epitope- The section on the antigen where the receptor binds is known as the epitope. Major Histocompatibility Complex- MHC molecules display fragments of intracellular proteins on the cell surface for immune surveillance. Understand the role of the physical organization of the immune system in its function. T cells are unable to recognise their own antigen unlike B-lymphocytes. Instead they rely on Professional Antigen Presenting Cells to present the antigen on their MHC and transport it to secondary lymphoid organs. Professional Antigen Presenting Cells Rise in body temperature coupled with increased production of a number of proteins by the liver: C-reactive protein Mannan-binding lectin erum-amyloid protein Collectively known as acute phase proteins/reactants C-reactive and serum-amyloid proteins bind to molecules on cell walls of bacteria and fungi Mannan-binding lectin binds to mannose sugar a carbohydrate not normally found on mammalian cells. These responses direct cells to phagocytose these cells. Cytokines- mall proteins that carry messages between cells. Can cause proliferation or activation of lymphocytes. Cells of the Innate Immune Response Granular leukocytes: 1. Macrophage- Phagocytic 2. Natural Killer Cells- identify and kill virus infected or tumour cells 3. Granulocytes a. Eosinophil- bi lobed b. Basophil- lobed c. Neutrophil- multi-lobed Neutrophil- Also called polymorphonuclear neutrophils because of the structure of their nucleus. Phagocytic and makes up the largest percentage of circulating leukocytes %. Eosinophil- Bi lobed nucleus and involved in response to parasitic infection around 1-3% of circulating leukocytes. Basophil- Not phagocytic, release granules containing, histamines, serotonins, prostaglandins, <1% important in T H2 response. Outline the role of clonal selection in immune responses. Within the body there is a large number of lymphocytes each with a specific antigen receptor as a result of random genetic recombination during their development. The cells that meet their specific antigen, proliferate and survive. A typical antigen is recognized by 1 in ~10 5 naive T cells. Most cells that have proliferated will die after they are no longer needed however a few remain as memory cells which causes the quicker secondary immune response. 1. Macrophage 2. B lymphocyte 3. Dendritic Cell Lymphoid Organs Primary Bone marrow Thymus econdary Lymph Nodes pleen Mucosa Associated Lymphoid Tissue (MALT) tuart's Immunology Page 3

4 Immune Cells and Organs 09 April :56 Learning Objectives Name the primary and secondary lymphoid organs and briefly differentiate between their functions. Draw simple diagrams to illustrate the structure of the thymus, lymph node, spleen, Peyer s patch and indicate the changes that occur after stimulation by antigen. Outline the recirculation of lymphocytes. Explain the use of CD (cluster of differentiation) markers for discrimination between lymphocytes. Compare and contrast phenotypic characteristics of B and T cells. Give examples of antigen presenting cells (APCs) and their locations. Outline the recirculation of lymphocytes. Naïve lymphocytes circulate in the blood and move to peripheral lymphoid tissue and then pass back to the blood and move throughout the blood. Eventually however lymphocytes either die or become activated. Name the primary and secondary lymphoid organs and briefly differentiate between their functions. Primary: Bone Marrow Thymus Primary lymphoid organs are lymphopoiesis and education occurs econdary: Mucosa associated lymphoid tissue pleen Lymph Nodes Where lymphocytes can interact with antigen other lymphocytes. Draw simple diagrams to illustrate the structure of the thymus, lymph node, spleen, Peyer s patch and indicate the changes that occur after stimulation by antigen. Thymus: Located in the anterior superior mediastinum at the level of about the 4th rib. Most t cells die in the thymus- deletion of autoreactive t cells- apoptosis Blood flow electin Integrin Bone marrow: Complicated network of capillaries within the bone involved in haematopoiesis. In a foetus blood cells are also made in the liver and the spleen, whereas in adults it is entirely the bone marrow of long flat bones. Another problem is how do the lymphocytes actually stop flowing through the blood and enter the tissues. HEV= High Endothelial Venule Venous sinuses Longitudinal artery inus 1. Rolling- This is the first stage and is influenced by selectin (CD62L) a molecule upon the surface of t cells which helps it slow down so it can enter the tissue. 2. Activation- binding of the t-lymphocyte to a chemokine tells it to move into the tissue. 3. Arrest/adhesion- The binding of Intergrin a form of ICAM halts the t cell so it can penetrate into the tissue. 4. Transendothelial migration Explain the use of CD (cluster of differentiation) markers for discrimination between lymphocytes. An internationally recognized systematic nomenclature for cell surface molecules. Used to discriminate between cells of the haematopoietic system. More than 300 CD markers. CD19 and 20 are present on all B cells. CD3 present on all T cells- transmits signal from TCR to nucleus. CD4+ and CD8+ = th and CTL. Longitudinal vein Bone Lymph Nodes: 1cm in size In via afferent out via efferent- A before E in alphabet. Lymphoid follicles are loose aggregations of B cells. Germinal centre- Centre of rapidly multiplying B lymphocytes. T cell area is inside of germinal centre and b cell area. Cortex= B area Paracortex= T area Compare and contrast phenotypic characteristics of B and T cells. T cell count= 1.5x10 9 per litre therefore 7.5x10 9 in blood. Blood contains only 2% of total pool. T-lymphocytes: T cells all express CD3. 90% have a αβtcr 2/3=CD4 1/3=CD8 10%=γδ TCR CD4+= T helper cells, regulatory T cells and they secrete cytokines. CD8+= Cytotoxic cell which lyse infected cells and secrete cytokines. Older people are more vulnerable to new flu strains as their thymic atrophy decreases the amount of new T cells that can be made to contain the infection. T cells only recognise processed antigen on MHC molecules. B lymphocytes: Produced by and develop in bone marrow. pleen: Acts as a filter for antigens in the blood. WHITE PULP= LYMPHOCYTE RED PULP= RED CELL tuart's Immunology Page 4

5 the amount of new T cells that can be made to contain the infection. T cells only recognise processed antigen on MHC molecules. B lymphocytes: Produced by and develop in bone marrow. urface antigen receptor: immunoglobulin like molecule. Express CD markers CD19 and CD 20. Express MHC Class II (can present antigen to helper T cells) Effector function is to produce antibodies. B cells recognise intact antigen free in body fluids i.e. not presented. They use the B cell receptor, a membrane anchored from of antibody linked to signaling subunits. Give examples of antigen presenting cells (APCs) and their locations. Antigen Presenting Cell- Cells that can present processed antigen to T lymphocytes to initiate an acquired (adaptive) immune response. 1. Dendritic cells- These are widely spread in the skin and mucosal tissue and present to T cells. Under the skin they are known as Langerhans cells. 2. Follicular dendritic cells- These are located in lymph node follicles and present to B cells. 3. Macrophage (activated)- Located in lymphoid tissue and present to T cells 4. B lymphocyte- Located in lymphoid tissue and present to T cells pleen: Acts as a filter for antigens in the blood. WHITE PULP= LYMPHOCYTE RED PULP= RED CELL Peri-arteriolar lymphatic sheath (PAL)= T cell area. Individuals who do not have a spleen are highly susceptible to infections with encapsulated bacteria. vein artery white pulp red pulp capsule Mucosa Associated Lymphoid Tissue M (microfold) cells are cells found in the follicle-associated epithelium of the Peyer's patch. They transport organisms and particles from the gut lumen to immune cells across the epithelial barrier, and are thus important in stimulating mucosal immunity. Unlike their neighbouring cells, they have the unique ability to take up antigen from the lumen of the small intestine via endocytosis or phagocytosis, and then deliver it via transcytosis to dendritic cells (an APC) and t-lymphocytes located in unique pocket-like structures on their basolateral side. Peyer's patch contains predominantly B lymphocytes and germinal centres during an immune response. They are aggregated lymphoid nodules. tuart's Immunology Page 5

6 Innate Immune ystem 09 April :55 Learning Objectives Briefly describe the functions of the important phagocytic cells: neutrophils, monocytes/macrophages. Define cytokines and describe their general properties. Define complement, list its major functions, and draw a simple diagram of the complement pathways. Describe a typical inflammatory response to a localised infection involving recruitment of neutrophils, and phagocytosis and killing of bacteria. Briefly describe the functions of the important phagocytic cells: neutrophils, monocytes/macrophages. Functions Phagocytosis: 1. Engulfment by pseudopodia which are finger like projections of the cell membrane. 2. Phagosome 3. Fusion to form phagolysosome 4. Killing, digestion 5. Waste release Briefly outline the events involved in a systemic acute phase response. Outline the phenotype and functions of natural killer (NK) cells. Define cytokines and describe their general properties. mall secreted proteins (70-80 aa long) whose function is in cell to cell communication. They generally act locally and have biological effects even at very low concentrations but however are short lived. Types of cytokine: 1. Interleukins- IL-x between leukocytes 2. Interferons- IFN: Anti-viral effects 3. Chemokines- Chemotaxis, movement 4. Growth factors 5. Cytotoxic- Tumor necrosis factor (TNF) ecrete Cytokines: Macrophages once activated secrete IL-12 and TNF which help activate Natural Killer cells. Natural killer cells secrete IFN- gamma which activates macrophages. Monocytes can also produce complement proteins. Neutrophil: hort lived cells that circulate in the blood then migrate into tissues. They are the first cells to be recruited to a site of tissue damage/infection. The larger denser primary granules are a sort of lysosome containing peroxidase, lysozyme and other hydrolytic enzymes. Macrophage: Less abundant, dispersed throughout the tissues, signal infection by release of soluble mediators. Important cytokines secreted by activated macrophages IL-1- Alarm cytokine, fever TNF-α- Alarm cytokine IL-6- Acute phase proteins, liver IL-8- Chemotactic for neutrophils IL-12- Directs adaptive immunity, activated NK cells. In order to fight infection neutrophils need to move to the site of infection, bind the pathogen, phagocytose and kill it. Chemotaxis and diapedesis Bacterial septic shock ystemic infection (gram negative bacteria) resulting from bacterial endotoxins cause a massive release of TNFα and IL-1 by activated macrophages. This results in an increased vascular permeability and a severe drop in blood pressure causing death in about 10% of cases. Dendritic cells also recognise pathogens and secrete cytokines, they take phagocytosed pathogens (antigens) to present at local lymph nodes. Define complement, list its major functions, and draw a simple diagram of the complement pathways. Major role in innate and antibody-mediated immunity. Complex series of ~30 proteins and glycoproteins, total serum conc. 3-4mg/ml Triggered enzyme cascade system which causes a rapid, highly amplified response. Components produced mainly in the liver, but also be monocytes and macrophages. There are 3 pathways: 1. Classical pathway Initiated by antigen-antibody complexes 2. Alternative pathway Direct activation by pathogen surfaces 3. Lectin pathway Antibody-independent activation of classical pathway by lectins which bind to Opsonisation- Coating of micro-organisms with proteins to facilitate phagocytosis Diapedesis- Movement of leukocytes through epithelial cells to get to site of infection Opsonins are molecules that bind to antigen, and also can be bound by phagocytes. Antibody and complement function as opsonins. Neutrophil Killing Mechanisms: Oxygen independent Enzymes Lysozyme Hydrolytic Antimicrobial peptides (defensins) Oxygen dependent Respiratory burst: Toxic metabolites uperoxide anion Hydrogen peroxide inglet oxygen Hydroxyl radical Reactive nitrogen intermediate tuart's Immunology Page 6

7 Initiated by antigen-antibody complexes 2. Alternative pathway Direct activation by pathogen surfaces 3. Lectin pathway Antibody-independent activation of classical pathway by lectins which bind to carbohydrates only found on pathogens. The classical and alternative pathways converge at C3b. C3 leads to the final common pathway. Ends with the formation of the Membrane Attack Complex (MAC) Classical Pathway Antigen/ Antibody The Y of Complement C1 C4b C2a Lectin Pathway MBL CRP C3b Pro-inflammatory C5 molecules C6,C7 C4a, C3a, C5a C8 C9 MAC Cell Lysis P D B Alternative Pathway Bacterium Principle Opsonin Enzymes Lysozyme Hydrolytic Antimicrobial peptides (defensins) uperoxide anion Hydrogen peroxide inglet oxygen Hydroxyl radical Reactive nitrogen intermediate Nitric oxide Phagocyte deficiency is associated with infections due to extracellular bacteria and fungi such as: taphylococcus aureus Pseudomonas aeruginosa Escherichia coli Candida albicans Aspergillus flavus Deep skin infections, impaired wound healing, poor response to antibiotics. Pro inflammatory molecules include C3, C4 and C5a C3b is the principle opsonin. Complement explanation Classical pathway- This depends on an antigen antibody interaction on the cell surface of a pathogen. The B cell binds to the antigen first, the C1 complex is then able to bind to the Fc region of the antibody once these complexes are brought close enough together. Then the complement cascade is initiated which involves C4a and C2a. C3b is then made which with c5,6,7,8,9 is able to form a membrane attack complex and lyse a bacteria. Alveolar macrophages in the lung. Kupffer cells in the liver. Microglial cells in the brain. Osteoclasts in bone Macrophage function Alternative pathway- A complement fragment C3 is broken down into C3b and C3a. C3b after reacting with proteins P, D and B is able to convert more of C3 into C3b and C3a which is a cascade mechanism and eventually allows the formation of an MAC. Lectin pathway- Complement is able to bind to carbohydrates on bacteria such as mannose binding lectin which with CRP causes the complement cascade. Control mechanisms: Lability (short half life) of components help achieve control. Likewise does the dilution of components in biological fluids. pecific regulatory proteins: Circulating, e.g. C1 inhibitor, Factor I, Factor H, C4 binding protein. Membrane bound e.g. CD59 interferes with MAC insertion, and DAF competes for C4b. Functions of complement include: lysis, opsonisation, inflammation and chemotaxis. Mast cells PAMP- TLR, RIG-I= viral genomes and NOD- like receptors (NLR) TLR7= ingle stranded RNA in viruses such as HIV + influenza TLR9= Double stranded DNA in bacteria + herpes simplex virus LP= Lipopolysaccharide Release of soluble mediators signal danger and infection. Describe a typical inflammatory response to a localised infection involving recruitment of neutrophils, and phagocytosis and killing of bacteria. ecrete histamine and other inflammatory mediators including cytokines. They are mucosal mast cells in the lungs. Connective tissue mast cell in the skin, peritoneal cavity and near blood vessels. They are able to recognise bacteria, phagocytose and kill them. They can be activated by complement products i.e. anaphylatoxins. Leads to vasodilation and an increase in vascular permeability. timulated by C3a and C5a. Outline the phenotype and functions of natural killer (NK) cells. They are large granulated lymphocytes which are cytotoxic, they lyse target cells and secrete IFN- γ. 5-10% of peripheral blood lymphocytes. No antigen specific receptor but express both activating and inhibitory receptors which Tissue damage resulting from bacterial infection will cause an inflammatory response. Firstly it can activate the alternative or classical pathways of complement activation. Complement can in turn release C3a and C5a which activates mast cells and causes them to degranulate and release prostaglandins, leukotrienes and histamine which increase vascular permeability. Furthermore the complement can opsonize the bacteria making it easier for tuart's Immunology Page 7

8 They are large granulated lymphocytes which are cytotoxic, they lyse target cells and secrete IFN- γ. 5-10% of peripheral blood lymphocytes. No antigen specific receptor but express both activating and inhibitory receptors which gives a balance of signals. They have receptors which bind to antibody coated cells (Antibody Dependent Cell Cytotoxicity) Important in defence against tumour cells and viral infections (esp. Herpes) Briefly outline the events involved in a systemic acute phase response. Local inflammatory response may be accompanied by a systemic response "acute phase" after 1-2 days. Fever, increased leukocytosis and production of acute phase proteins in the liver: i. erum amyloid protein ii. C-reactive protein C polysaccharide of pneumococcus Activates complement and levels may increase 1000 fold. iii. Mannan binding lectin protein Opsonin for monocytes and activates complement iv. Complement v. Fibrinogen (clotting) Firstly it can activate the alternative or classical pathways of complement activation. Complement can in turn release C3a and C5a which activates mast cells and causes them to degranulate and release prostaglandins, leukotrienes and histamine which increase vascular permeability. Furthermore the complement can opsonize the bacteria making it easier for macrophages to phagocytose them. They in turn will become activated and release cytokines IL-1 and IL-6 which are pro-inflammatory and IL-8 which will act as a chemoattractant for neutrophils to enter the tissues. imilarly complement can also act as a chemoattractant for neutrophils. Tissue damage can result in endothelial injury causing plasmin release and the initiation of the blood clotting cascade ending with formation of fibrin. Furthermore bradykinin can be released which increases vascular permeability and lowers blood pressure. Other leukocytes also reach the area and stop via action of selectin, integrins and chemokines and force their way into the tissue. tuart's Immunology Page 8

9 Antibodies 10 April :37 Learning Objectives Describe with the aid of a simple diagram the immunoglobulin molecule, identifying the antigen-binding site (Fab) and Fc portions of the molecule. Briefly describe the properties of the antigen-binding site. Distinguish between antibody affinity and avidity. List the immunoglobulin classes and sub-classes in man. Describe their functions and relate these to their individual structure. Briefly describe the properties of the antigen-binding site. The fragment antigen binding site is composed of one constant and one variable domain of both the heavy and light chains. Which is at the amino end of the terminal. Of the variable heavy domain (V H) there are 3 hypervariable regions known as CDR1, 2 and 3, where CDR stands for Complementary determining regions. Describe with the aid of a simple diagram the immunoglobulin molecule, identifying the antigen-binding site (Fab) and Fc portions of the molecule. Each individual has >10 7 different antibodies, with secondary effector functions including complement activation, opsonisation and cell activation via specific binding receptors (Fc receptors) N N N N L H H L H = HEAVY CHAIN (e.g. 50kDa) both the same L = LIGHT CHAIN (25kDa) both the same N, C = POLYPEPTIDE TERMINAL C C = DIULPHIDE BRIDGE C C The heavy chain is about 50kDa in size and are the two longer lines in the middle. The light chain is 25kDa in size and is on the outside. The N (amino terminal) is on the left of immunoglobulin and the carboxy terminus is on the right. Joining the chains to each other are disulphide bridges which indicate cysteine amino acid residues. Portion that is just heavy chain on the right is the Fc. Fab and Fc Limited digestion of gamma globulin by purified papain resulted in 3 fragments each 50kDa in size. 2 fragments has antigen binding activity Fab and the other did not and formed protein crystals Fc. Distinguish between antibody affinity and avidity. Antibody affinity- The strength of the total non-covalent interactions between a single antigen binding site and a single epitope on the antigen. Antibody avidity- The overall strength of multiple interactions between an antibody with multiple binding sites and a complex antigen with multiple epitopes List the immunoglobulin classes and sub-classes in man. Describe their functions and relate these to their individual structure. Antibody cross reactivity: Vaccination with cowpox induces antibodies which are able to recognise smallpox. ABO blood-group antigens (glycoproteins on R.B.C) Antibodies made against microbial antigens on common intestinal bacteria may crossreact with carbohydrates on R.B.C. Isotypes vs allelotypes Isotypes are present in everybody kappa, lambda, heavy chains, classes and subclasses. Allelotypes are allelic polymorphisms which vary in the population e.g. gamma chain. CLA HEAVY CHAIN Flexibility of antibody molecules C H DOMAIN tuart's Immunology Page 9

10 CLA HEAVY CHAIN C H DOMAIN LIGHT CHAIN IgG IgA IgM IgD IgE Flexibility of antibody molecules List the immunoglobulin classes and sub-classes in man. Describe their functions and relate these to their individual structure. IgG Both light and heavy chains can be divide into variable regions and constant regions. Gamma heavy chain and is the most abundant immunoglobulin. It occurs as a monomer but has 4 sub classes and variability is mainly located in hinge region and effector function domains. It is actively transported across the placenta. It is present in blood and extracellular fluids. It is a major activator of classical complement pathway- mainly IgG1 and 3. Ig88 List the immunoglobulin classes and sub-classes in man. Describe their functions and relate these to their individual structure. IgD Delta heavy chain and is present in extremely low serum levels. It is the least well characterised immunoglobulin and surface IgD is expressed early in B cell development which indicates its important there, it is also involved in their activation. Epsilon heavy chain once again very low serum levels. It is produced in response to parasitic infections and in allergic disease. It also binds to high affinity Fc receptors of mast cells and basophils. Cross linking by antigen triggers mast cell activation and histamine release. IgE IgG1 is the most abundant at 70%. IgG3 activates complement the most. 1 and 4 vs tetanus, 2 vs bacterial polysaccharides. IgA Alpha heavy chain. econd most abundant after IgG. It occurs as a monomer in blood and dimer in secretions. It is a major secretory immunoglobulin that protects mucosal surfaces from bacteria, viruses and protozoa. IgM Mu heavy chain and is a large pentameric molecule which is formed by 5 monomers joined by a J chain. (10x Fab) 80% is confined to the blood (mainly due to size), and it is the first Ig synthesised after exposure to an antigen. Its multiple binding sites compensate for low affinity and makes it efficient at agglutination, it also activates complement. elective Ig distribution IgG and M in blood. IgG in extracellular fluid. Dimeric IgA in breast milk and secretions across epithelia. Maternal IgG in foetus via placenta. IgE with mast cells below epithelium. Brain devoid of Igs. tuart's Immunology Page 10

11 Maternal IgG in foetus via placenta. IgE with mast cells below epithelium. Brain devoid of Igs. GAMED is decreasing order of abundance in the body. tuart's Immunology Page 11

12 B lymphocytes 10 April :04 Learning Objectives Briefly outline the principles of immunoglobulin (Ig) gene rearrangement in the generation of diversity Describe the process of stimulation of individual B cells to divide and secrete antibody such as to generate immunity to particular antigen (clonal selection) Outline the differences in antibody production during primary and secondary immune responses B cell development and receptor structure Adaptive Immune Response: This occurs 4-7 days after encounter of an antigen and needs time to develop. B cells are the humoral response and produce antibodies and t cells are the cell mediated response that lyses cells and produced cytokines. B cell development Differentiate between monoclonal and polyclonal antibody Describe the process of stimulation of individual B cells to divide and secrete antibody such as to generate immunity to particular antigen (clonal selection) Briefly outline the principles of immunoglobulin (Ig) gene rearrangement in the generation of diversity There are different antibody molecules that can be generated. Each antibody is produced by a B lymphocyte expressing a specific BCR. Antigen Receptor Diversity Functional genes for antigen receptors do not exist until they are generated during lymphocyte development. Each BCR receptor chain (κ,λ and heavy chain genes) is encoded by separate multigene families on different chromosomes. During B cell maturation these gene segments are rearranged and brought together. This process is called Immunoglobulin gene rearrangement. Immunoglobulin gene rearrangement generates the diversity of the lymphocyte repertoire. Lamda chain located on chromosome 22 Kappa chain located on chromosome 2 Heavy chains located on chromosome Interaction between stromal cells and precursor B cells via VCAM-1, VLA-4 and CAMs. 2. Upregulation of CD117 (Kit) and binding to CF- tem cell factor 3. Pro-b cells proliferate 4. Expression of pre-b cell receptor 5. B-lineage cells move towards the central axis of the marrow cavity. tructure of B cell receptor Transmembrane protein complex composed of mig and di-sulfate linked heterodimers, IgA, IgB. IgA/IgB heterodimers contain immunoglobulin fold structure. The cytoplasmic tail of mig is too short to signal. The cytoplasmic tails of IgA and B are long enough to interact with intracellular signalling molecules. The B cell receptor is made before the cell ever encounters an antigen and is present in thousands of identical copies on the surface of the B-lymphocyte. Describe the process of stimulation of individual B cells to divide and secrete antibody such as to generate immunity to particular antigen (clonal selection) election for self tolerance + productive gene rearrangement, if the cells survive, they will become mature (or naïve) B cells (IgM and IgD). Although naïve and mature seem opposites that mean the same thing in that the cell has not encountered antigen yet. IL-7 expressed in early stages which is a growth factor receptor. Outline the differences in antibody production during primary and secondary immune responses Each lymphocyte (T or B) expresses an antigen receptor with a unique specificity. Binding of antigen to its specific receptor leads to activation of the cell, causing it to proliferate into a clone of cells. All of these clonally expanded cells bear receptors of the same specificity to the parental cell. Lymphocytes expressing receptors that recognise self molecules are deleted early during lymphocyte development. Activation of B cells: tuart's Immunology Page 12

13 parental cell. Lymphocytes expressing receptors that recognise self molecules are deleted early during lymphocyte development. Activation of B cells: Naïve antigen specific lymphocytes cannot be activated by antigen alone. B cells require an accessory signal: a. Directly from microbial constituents Thymus independent- Only IgM has no memory b. From a T helper cell. Thymus dependent- all Ig classes and has a memory. Antibody production response to Thymus-dependent antigens. Antigen recognition and presentation 1) The membrane bound BCR recognises antigen 2) The receptor-bound antigen is internalised and delivered to intracellular sites 3) The internalised antigen is degraded into peptides 4) Peptides associate with self molecules (MHC class II) 5) The antigen/self-complex is expressed at the cell surface T lymphocytes with a complementary antigen receptor recognise the complex T helper cells secrete lymphokines after recognition of the antigenic/self complex on the surface of the B cell. B cell enters into the cell cycle, divides and develops into a clone of cells with identical BCRs. B cells differentiate into plasma cells secreting these soluble BCRs= antibodies. B cells differentiate into memory cells. T-B cell collaboration Differentiate between monoclonal and polyclonal antibody Invading microorganisms have multiple antigenic epitopes- a mixture of antibodies directed to several antigenic determinants will be produced which are derived from many different clones of B cells= a polyclonal response. Monoclonal antibodies are derived from a single B cell clone. Myeloma- A cancerous plasma cell that divides permanently without antigenic stimulation and secretes antibodies which are indistinguishable from normal antibody= myeloma proteins. Antigen cross linking induces signal 1 which cause MHC II and B7 to increase. Internalisation and presentation of antigen results in T helper cell recognising the antigen and co-stimulation occurring resulting in activation of T cells. Activated T cell expresses CD40L. Interaction between CD40L and CD40 on B cell provides the second activation signal for the B cell. B7- CD28 interaction provides co-stimulation for Th cells. Activated B cells express cytokine receptor for T cell derived cytokines which cause them to proliferate and differentiate into plasma cells. Plasmacytoma- Clone of malignant plasma cells. B cell decisions: These are the result of any integrated signals which determine the quality and quantity of antibody produced. Choice between proliferation or cell death of perifollicular B cells occurs in the first 3 days after antigen exposure. Perifollicular B cells differentiate into extrafollicular plasma cells or germinal centre B cells. Positive selection of BCR affinity for foreign antigen versus negative selection for self antigen in germinal centre B cells. urvival versus death of antibody secreting plasma cells. Cytokines and class switching: IL-4 induces IgG1 and IgE. tuart's Immunology Page 13

14 IL-5 augments production of IgA. IFN-gamma induces IgG3 and IgG2a. TGF-beta induces IgG2b and IgA. tuart's Immunology Page 14

15 T lymphocytes 10 April :14 Learning Objectives Outline the origins and functions of T lymphocyte subsets. Briefly describe the structure and distribution of major histocompatibility complex (MHC) class I and class II molecules. Outline the origins and functions of T lymphocyte subsets. T-lymphocytes destroy intracellular pathogens. It detects foreign antigens via the TCR. Antigen seen is presented by MHC molecule on the surface of host infected cell. Outline the mechanisms by which antigen presenting cells (APCs) process and present antigens. Compare and contrast antigen recognition by B and T lymphocytes and by CD4+ and CD8+ T lymphocytes. Briefly describe the structure and distribution of major histocompatibility complex (MHC) class I and class II molecules. The MHC is a group of tightly linked genes important in specific immune responses. MHC molecules are found in all vertebrates and can present antigens to T lymphocytes. Each type has a: 1. Peptide binding 2. Immunoglobulin like 3. Transmembrane 4. Cytoplasmic regions carbohydrate variable region (V) constant region (C) (hinge) transmembrane A small subset of T cells use gamma and delta chains. ~90aa N N -microglobulin C ~25aa ~30aa C ~90aa N N ~90aa ~90aa MHC CLA I PEPTIDE BINDING REGION IMMUNOGLOBULIN LIKE REGION TRANMEMBRANE REGION CYTOPLAMIC REGION ~90aa MHC CLA II PEPTIDE BINDING REGION disulfide bond cytoplasmic tail There are 4 possible variable regions alpha, beta, gamma and delta. Alpha and beta are much more common. CD3+ which all T cells express is a signalling region composed of (gamma, delta, epsilon and zeta regions) which allows signals caused by TCR to be passed onto the nucleus for gene action. CD4+ co-receptor cells (TH) see peptide on MHC class II CD8+ (cytotoxic) see peptide on MHC Class I Co-receptor molecules bind to the relevant MHC, increase the avidity of T cell-target interaction and are important in signalling. CD8 induce apoptosis in the target cell and also secrete cytokines. CD4 helper t cells secrete cytokines and recruit effector cells of innate immunity and help activate macrophages. They also amplify and help T and B cell responses. T lymphocytes only recognise processed antigens displayed on cell surfaces. CD8 + CTL CD4 + TH1 and TH2 ~90aa ~90aa IMMUNOGLOBULIN LIKE REGION ~25aa TRANMEMBRANE REGION variable length C C CYTOPLAMIC REGION Details MHC I MHC II Peptide and immunoglobulin regions Trans membrane region Cytoplasmic region Peptide length α1,2,3 and β2 microglobulin Way to remember is the there is an odd one out. Each is 90 aa long. 1x 25aa long TMR α1,2 and β1,2. Way to remember II of each. Each is 90 aa long. 2 x 25aa long TMR 30 aa long. Variable length. Accommodates small peptides 8-10 aa long. Genes B, C and A DP, DQ and DR MHC Class I only bind with alpha chains. MHC Class II has two transmembrane domains. HUMAN LEUKOCYTE ANTIGEN Accommodates larger peptides >13aa long. In pic there is gap between nitrogens. The Thymus + Development Important in T cell maturation- immature thymocytes are in the cortex, mature thymocytes migrate to the medulla. Immature thymocytes are CD4- and CD8- which means they display neither. In the cortex the t cells only express a pretcr+ which is composed of beta and surrogate alpha TCR. There is a stage at which the thymocytes display CD4+CD8+TCR+. In medulla they have developed fully and have only TCR and CD4+/CD8+ not both. Huge variety of TCRs made by gene rearrangements/recombination. Thymus checks if the new beta chain is functional and if the whole of the αβtcr is functional and if it is dangerous/autoreactive. Only if these tests are passed can the t cell continue to develop. Useless- means that they can't see MHC and thus they die by apoptosis. Useful actually means that they see MHC weakly this is the criteria for positive selection. Dangerous means that they have too high an affinity for MHC and see it strongly and thus they receive a signal to die by apoptosis. Only 5%of thymocytes survive selection. DP A B Class II DQ A B DR A B tuart's Immunology Page 15 Class I B C A Genes Cell membrane Outline the mechanisms by which antigen presenting cells (APCs) process and present antigens.

16 HUMAN LEUKOCYTE ANTIGEN DP A B Class II DQ A B DR A B Class I B C A Genes Cell membrane Outline the mechanisms by which antigen presenting cells (APCs) process and present antigens. APC phagocytose or ingest the antigen, sometimes it is chopped up/processed and is then presented on MHC class II molecules. HLA-A HLA-DP HLA-DQ HLA-DR HLA-B HLA-C CLA I As you can see above there are 6 different genes responsible for MHC/HLA phenotype. MHC Gene Expression Not just MHC class I and II. 3.6 million base DNA sequence published in functional genes, 40% of which have immune related function. The MHC is polygenic which means there are several class I and II loci. Expression is co-dominant (maternal and paternal genes are both expressed). MHC Class I: Nearly all nucleated cells, although at various levels. Levels may be altered during infection, or by cytokines. MHC Class II: Normally only on professional antigen presenting cells and again may be regulated by cytokines. Polymorphism: Human MHC genes are highly polymorphic large number of alternative different versions of the same gene within the population. Each version is termed an allele. MHC haplotype- A group of MHC alleles linked on one chromosome Compare and contrast antigen recognition by B and T lymphocytes and by CD4+ and CD8+ T lymphocytes. TAP TRANPORTER AOCIATED WITH ANTIGEN PROCEING Class I: Memory aide- You'd wanna "TAP" 1st class bitches. a. This is where the antigen (protein) is already present within a cell because it is infected. The proteosomes degrade the proteins whereby it becomes transported by Transporter Associated with antigen Processing (TAP). b. This peptide is then assisted in its folding by the chaperone molecule Calnexin in the RER to become a MHC Class I molecule and then is transported to the Golgi (probably modified further) and is secreted sent to the cell membrane for display. Class II: This is the pathway involving exogenous antigen. a. The antigen is endocytosed into a vesicle and displayed on Class II MHC molecules. b. Class II MHC are produced in the RER with an attached Invariant chain which inactivates it. This chain is digested by Class II associated Invariant chain Peptide (CLIP) which allows the binding of the antigen to the MHC Class II molecule and its presentation on the cell surface. Antigens in different locations require different responses. Different pathways present antigen from different locations to different T cell sub sets. Endogenous AG (cytoplasm) to Class-I restricted CD8 T cells. Exogenous AG (external) to Class II-restricted CD4 T cells. B lymphocytes are able to directly recognise intact, extracellular antigen whereas t lymphocytes can only recognise processed antigens. Cytotoxic= MHC Class I and Helper T Cells= MHC class II. tuart's Immunology Page 16

17 Effector T-Lymphocytes 12 April :09 Learning Objectives Explain the different requirements for activation of naïve and memory T lymphocytes Describe effector functions of T lymphocytes including cell-mediated cytotoxicity, macrophage activation, delayed type hypersensitivity and T/B lymphocyte cooperation Briefly outline the function of T helper cells in relation to the cytokines they produce Outline the importance of antigen presenting cells in the induction of T lymphocyte responses Explain the different requirements for activation of naïve and memory T lymphocytes The role of T-cell mediated immunity is to detect and eliminate intra-cellular pathogens and altered cells such as tumour cells. Naïve T cells need 2 signals to be activate: i. Antigen recognition by TCR ii. Co-stimulation signal Antigen recognition in the absence of co-stimulation leads to tolerance in peripheral T cells. Effector (memory?) T cells are less dependent upon co-stimulation and are able to kill just be pathogen recognition. CD80 to CD28= APC to T cell activation Describe effector functions of T lymphocytes including cell-mediated cytotoxicity, macrophage activation, delayed type hypersensitivity and T/B lymphocyte cooperation Proliferation after activation Generation of cytotoxic T lymphocytes Resting T cells only express gamma and beta chains of IL-2 receptor. Activated T cells express gamma, beta and alpha chains and secrete IL-2. Naïve T cells: mature recirculating T cells that have not yet encountered antigen Effector T cells: encountered antigen, proliferated and differentiated into cells that participate in the host defense Explanation: APC present antigen on a class II MHC to TH1 helper cells. TH1 proliferate by producing IL-2. Cytotoxic t lymphocyte precursor (CTLP) are activated by binding antigen on Class I MHC to its TCR, and via a co-stimulatory signal and thus they become Ag-activated CTL-P. IL-2 produced by Th1 cells causes Ag-activated CTL-P to proliferate and differentiate into the fully fledged cytotoxic T lymphocytes. Cell-mediated cytotoxicity Target cells: Cells on which effector T cells act Describe effector functions of T lymphocytes including cell-mediated cytotoxicity, macrophage activation, delayed type hypersensitivity and T/B lymphocyte cooperation Delayed Type Hypersensitivity Can be protective as well as pathological, primary role in defence against intracellular pathogens. If source of antigen is not eradicated then chronic inflammation and granuloma formation tuart's Immunology Page 17

18 IL-2 produced by Th1 cells causes Ag-activated CTL-P to proliferate and differentiate into the fully fledged cytotoxic T lymphocytes. Cell-mediated cytotoxicity Binding of antigenic peptide from MHC Class I molecules to TCR causes polarisation of the cytoskeleton and cytoplasmic components so that lytic granules can be secreted to the pole in contact with the infected cell. Apoptosis is characterised by fragmentation of nuclear DNA. CTLs also release IFN-gamma- which inhibits viral replication and activates macrophages. TNF alpha and beta synergise with IFN-gamma receptors. Delayed Type Hypersensitivity Can be protective as well as pathological, primary role in defence against intracellular pathogens. If source of antigen is not eradicated then chronic inflammation and granuloma formation occurs. If the antigen is not a microbe, like in the CH (chronic hyperplastive sinusitis?), the DTH produces tissue injury without protection= Hypersensitivity Consists of 2 phases 1. ensitization phase APC presents antigen and causes TH1 cells or CD8+ cells to develop into T DTH 2. Effector Phase TDTH cells secrete various cytokines including IFN-γ and TNFβ. These in turn cause macrophages to become activated which increases expression of MHC class II molecules, TNF receptors and use of oxygen. Granules and Fas-FasL interactions. Granule: 1. The granule secreted by the CTL contains perforin which polymerizes to form a pore within the membrane. 2. Granzymes pass through, and via a Caspase Cascade are able to induce apoptosis. Fas pathway: 1. Fas ligand on CTL binds to Fas receptor on the infected cell. 2. Fas Associated Death Domain Protein (FADD) is produced with an initiator caspase which go on together to initiate apoptosis via a cascade mechanism. CTLs can be reused. Memory Cells CD45RA expression allows to differentiate between naïve and memory cells. Expression of the chemokine receptor CCR7, which controls homing to secondary lymphoid organs, allows a further subdivision of human memory cells. There are two subsets of memory T cells: i. CCR7-CD45RA-memory cells= Effector Memory cells= TEM These display immediate effector function ii. CCR7+CD45RA-memory cells= Central memory cells= TCM These lack immediate effector function, they differentiate into CCR7- effector cells after secondary stimulation. Outline the importance of antigen presenting cells in the induction of T lymphocyte responses Antigen presenting cells such as activated dendritic cells, activated B lymphocytes and activated macrophages are necessary to activate T lymphocytes. If T lymphocytes are not activated then they cannot become effector T lymphocytes. For CTLs antigen presenting cells i.e. MHC Class II would appear not to be necessary as CTLs recognise MHC Class I. However APC activate TH cells which then produce a variety of cytokines including IL-2 which allows the full development and proliferation of CTLs. Regulatory T cells T cells that regulate the activation or effector functions of other t cells. They are necessary to maintain tolerance to self antigens. They can inhibit the activation of macrophages by TH1 cells which is a regulatory function. Briefly outline the function of T helper cells in relation to the cytokines they produce CD4+ T cells are critical in host defence (HIV as example) and naïve CD4+ can differentiate into distinct subsets after antigen encounter. Produce restricted cytokine patterns. ubtypes Follicular helper T cells (Tfh): Reside in B cell follicles Essential for generation of isotype-switched antibodies and in B cell memory. Th9 cells: how in vitro, in vivo relevance unclear. tuart's Immunology Page 18